Method and kit for detection of anti-zika virus antibodies

ABSTRACT

Provided is a method of detecting the presence of an anti-Zika virus (ZIKV) antibody in a sample, including contacting a sample with a suspension having a plurality of microspheres wherein individual microspheres are conjugated to a peptide and the peptide includes a ZIKV peptide selected from the group including ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, forming a first incubated suspension by incubating said sample with said suspension to permit binding of anti-ZIKV antibodies present in the sample to said microspheres, forming a second incubated suspension by contacting said first incubated suspension with an anti-ZIKV antibody detecting-reagent to permit binding of the anti-ZIKV antibody detecting reagent to said microspheres, removing from the second incubated suspension anti-ZIKV antibody detecting-reagent molecules that are not bound to said microspheres, and detecting the presence of anti-ZIKV antibody detecting-reagent molecules in the second incubated suspension. Also provided is a kit containing reagents and compositions for performing the foregoing method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application No.62/421,694, filed Nov. 14, 2016, and this application claims priority toU.S. provisional application No. 62/438,681, filed Dec. 23, 2016, thecontents of which applications are incorporated by reference in theirentireties into the present disclosure.

FIELD OF THE INVENTION

The present disclosure relates to, inter alia, methods for detection ofZika virus infection and compositions of matter and kits for use inperforming said method. More particularly, methods for detection ofanti-Zika virus antibodies is provided. More particularly still, amethod for detecting the presence of anti-Zika virus antibodies, anddistinguished from the presence of antibodies to other flaviviruses.Also disclosed are kits for the performance of such methods.

BACKGROUND OF THE INVENTION

Zika virus (ZIKV) belongs to the genus of flavivirus within the familyFlaviviridae. Many flaviviruses are significant human pathogens,including ZIKV, yellow fever (YFV), dengue virus (DENV serotypes 1 to4), Japanese encephalitis virus (JEV), West Nile virus (WNV), andtick-borne encephalitis virus (TBEV). ZIKV is predominantly transmittedby Aedes spp. mosquitoes, which also transmit DENV and YFV, as well aschikungunya virus (an emerging alphavirus). Besides mosquitoes, ZIKV canalso be transmitted through maternofetal route, sex, blood transfusion,and organ transplantation. Approximately 80% of the ZIKV infections areasymptomatic. Disease symptoms associated with ZIKV infection includeheadaches, fever, lethargy, rash, conjunctivitis, myalgia, andarthralgia. Severe diseases of ZIKV infection include neurotropicGuillain-Barre syndrome and congenital microcephaly. The flavivirusgenome is a single-strand, positive-sense RNA of approximately 11,000nucleotides. It contains a 5′ untranslated region (UTR), an open-readingframe (ORF), and a 3′ UTR. The single ORF encodes a long polyproteinwhich is processed into ten viral proteins, including three structuralproteins—capsid (C), precursor membrane (prM), and envelope (E)—andseven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, andNS5).

Diagnosis of ZIKV infection is performed through detection of viralcomponents (e.g., viral RNA, viral proteins, or virus isolation) anddetection of host immune response (e.g., antibodies against viralproteins). For viral component-based diagnosis, RT-PCR, immunoassay, andvirus isolation detect ZIKV RNA, viral proteins, and live virus,respectively (Lanciotti et al., 2008); among them, RT-PCR is the mostpopular assay because of its sensitivity and specificity. The viremicphase of ZIKV infection usually lasts for about one week, yetoccasionally persists beyond two weeks. Due to the short duration of theviremic phase, the diagnostic window for detection of viral componentsis narrow. Therefore, host immune response-based assays play animportant role, among which enzyme-linked immunosorbent assays (ELISA),such as IgM-capture ELISA (MAC-ELISA), and plaque reductionneutralization test (PRNT) are the two most commonly used serologicassays in ZIKV diagnosis. Conventionally, serologic diagnosis of Zikavirus (ZIKV) infection relies mainly upon IgM-capture ELISA which isconfounded with the flaw of cross-reactivity among differentflaviviruses. Unfortunately, the interpretation of conventionalIgM-capture ELISA assays for ZIKV and other flaviviruses are challengingdue to the cross-reactive nature of anti-flaviviral antibodiesconventionally used in such tests, leading to equivocal diagnosticresults. This challenge is confounding Zika diagnosis because (i) manyflaviviruses (e.g., ZIKV and DENV) produce similar disease symptoms and(ii) antibodies from patients infected with ZIKV cross-react with otherflaviviruses. Consequently, ZIKV IgM-capture ELISA results typicallyrequire neutralization tests for confirmation. Furthermore, PRNT istime-consuming, labor-intensive, slow, and low-throughput, andcost-ineffective, impairing attempts at rapid diagnosis to halt or slowspread if infection. Moreover, PRNT still relies upon bothvirus-specific and cross-reactive epitopes of viral E protein such thatthe results may be inconclusive with respect to flavivirus infections(Shan et al., 2016a). There is therefore a need to improve the accuracyand speed of serologic diagnosis for flaviviruses, ZIKV in particular.

SUMMARY OF THE INVENTION

The present disclosure relates to, inter alia, a method of detecting thepresence of an anti-Zika virus (ZIKV) antibody in a sample, includingcontacting a sample with a suspension having a plurality of microsphereswherein individual microspheres are conjugated to a peptide and thepeptide includes a ZIKV peptide selected from the group including ZIKVNS1, ZIKV NS5, and ZIKV envelope protein, forming a first incubatedsuspension wherein forming includes incubating said sample with saidsuspension to permit binding of anti-ZIKV antibodies present in thesample to said microspheres, forming a second incubated suspensionwherein forming includes contacting said first incubated suspension withan anti-ZIKV antibody detecting-reagent to permit binding of theanti-ZIKV antibody detecting reagent to said microspheres, removing fromthe second incubated suspension anti-ZIKV antibody detecting-reagentmolecules that are not bound to said microspheres, and detecting thepresence of anti-ZIKV antibody detecting-reagent molecules in the secondincubated suspension.

In some embodiments, the method includes individual microspheresconjugated to ZIKV peptides that differ from ZIKV peptides to whichother individual microspheres are conjugated, and the ZIKV peptides areselected from ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, and any twoof the foregoing.

In other embodiments, different ZIKV peptides are ZIKV NS1 and ZIKV NS5.In some embodiments, individual microspheres are conjugated to ZIKV NS1,and in other embodiments individual microspheres are conjugated to ZIKVNS5. In some embodiments, some microspheres are conjugated to ZIKVEnvelope protein, some microspheres are conjugated to ZIKV NS1, and somemicrospheres are conjugated to ZIKV NS5. In further embodiments, somemicrospheres are conjugated to ZIKV Envelope protein and somemicrospheres are conjugated to ZIKV NS1.

In other embodiments, the sample includes a biological sample from asubject and the biological sample is selected from the group includingbodily fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous,tissue, tissue homogenate, cellular extract, spinal fluid, and anycombination of two or more of the foregoing. In other embodiments, thesubject is a human.

In other embodiments, detecting includes measuring an autofluorescencesignal emitted by an anti-ZIKV antibody-detecting reagent, anautofluorescence signal emitted by a microsphere, or an autofluorescencesignal emitted by an anti-ZIKV antibody-detecting reagent and anautofluorescence signal emitted by a microsphere.

Some embodiments further include detecting the presence of an antibodyto a Dengue virus (DENV) protein in a sample, wherein the proteincomprises NS1 and the DENV comprises DENV1, DENV2, DENV3, DENV4, or anycombination of two or more of the foregoing. For example, somemicrospheres may be conjugated to DENV1 NS1, some microspheres may beconjugated to DENV2 NS1, some microspheres may be conjugated to DENV3NS1, and some microspheres may be conjugated to DENV4 NS1. In furtherembodiments, some microspheres may be conjugated to ZIKV Envelopeprotein, some microspheres may be conjugated to ZIKV NS1, somemicrospheres may be conjugated to ZIKV NS5, some microspheres may beconjugated to DENV1 NS1, some microspheres may be conjugated to DENV2NS1, some microspheres may be conjugated to DENV3 NS1, and somemicrospheres may be conjugated to DENV4 NS1. In still furtherembodiments, some microspheres may be conjugated to ZIKV Envelopeprotein, some microspheres may be conjugated to ZIKV NS1, somemicrospheres may be conjugated to DENV1 NS1, some microspheres may beconjugated to DENV2 NS1, some microspheres may be conjugated to DENV3NS1, and some microspheres may be conjugated to DENV4 NS1.

In another aspect, provided is a kit for detecting the presence of ananti-Zika virus (ZIKV) antibody in a sample, including a plurality ofmicrospheres wherein individual microspheres are conjugated to a peptideand the peptide comprises a ZIKV peptide selected from the groupconsisting of ZIKV NS1, ZIKV NS5, and ZIKV envelope protein. In someembodiments, the kit includes individual microspheres conjugated to oneof two or three different ZIKV peptides which differ from ZIKV peptidesto which other microspheres are conjugated to, and the different ZIKVpeptides are ZIKV NS1, ZIKV NS5, and ZIKV envelope protein, or any twoof the foregoing. In some embodiments, the different ZIKV peptides areZIKV NS1 and ZIKV NS5. In other embodiments, the different ZIKV peptidesare ZIKV envelope protein, ZIKV NS1, and ZIKV NS5. In furtherembodiments, the different ZIKV peptides are ZIKV envelope protein andZIKV NS1. In still further embodiments, the kit may further includeindividual microspheres conjugated to one of two or more different DENVpeptides which differ from DENV peptides to which other microspheres areconjugated to, and the different DENV peptides are DENV1 NS1, DENV2 NS1,DENV3, NS1, or DENV4 NS1. For example, a kit may include microsphereswith the following peptides conjugated thereto: ZIKV NS1, ZIKV NS5, ZIKVEnvelope protein, DENV1 NS1, DENV2 NS2, DENV3 NS3, and DENV4 NS4. Insome examples, a kit may contain a subset of the foregoing. For example,a kit may contain microspheres with the following peptides conjugatedthereto: ZIKV NS1, ZIKV Envelope protein, DENV1 NS1, DENV2 NS2, DENV3NS3, and DENV4 NS1. In yet other embodiments, a kit may contain anycombination of any two or more of the foregoing microspheres. Forexample, in some embodiments, the different ZIKV peptides are ZIKV NS1and ZIKV NS5. In other embodiments, the different ZIKV peptides are ZIKVenvelope protein, ZIKV NS1, and ZIKV NS5. In further embodiments, thedifferent ZIKV peptides are ZIKV Envelope protein and ZIKV NS1.

In other embodiments, individual microspheres are conjugated to ZIKVNS1. In still other embodiments, individual microspheres are conjugatedto ZIKV NS5. In still other embodiments, the kit includes an anti-ZIKVantibody-detecting reagent.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating aspects of the present invention, thereare depicted in the drawings certain embodiments of the invention.However, the invention is not limited to the precise arrangements andinstrumentalities of the embodiments depicted in the drawings. Further,as provided, like reference numerals contained in the drawings are meantto identify similar or identical elements. The foregoing and otherobjects, features, and advantages of the invention are apparent from thefollowing detailed description taken in conjunction with theaccompanying drawings in which:

FIG. 1 shows SDS-PAGE analysis of recombinant ZIKV NS5 protein.Full-length NS5 of ZIKV was expressed in an E. coli system, and purifiedthrough affinity followed by size-exclusion chromatography (see detailsin Materials and Methods). The recombinant protein was analyzed on a 12%Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad).

FIG. 2 shows a Table (Table 5) of a summary of PRNT, IgM-capture ELISA,and multiplex MIA diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of an invention disclosed herein and certain features,advantages, and details thereof, are explained more fully below withreference to the non-limiting embodiments illustrated in theaccompanying drawings. Descriptions of well-known materials, fabricationtools, processing techniques, etc., are omitted so as to notunnecessarily obscure the invention in detail. It should be understood,however, that the detailed description and the specific examples, whileindicating some embodiments, are given by way of illustration only, andare not by way of limitation. Various substitutions, modifications,additions and/or arrangements within the spirit and/or scope of theunderlying inventive concepts will be apparent to those skilled in theart from this disclosure.

Disclosed herein is a method for detecting the presence of anti-ZIKVantibodies in a sample, overcoming shortcomings of the prior art.Conventionally, detection of anti-ZIKV antibodies in a sample mayrequire a high volume of sample, may take longer than several hours toperform, may be of low sensitivity resulting in a failure to detect apresence of anti-ZIKV antibodies in a sample, and low selectivityresulting in evidence of a presence of anti-ZIKV antibodies in a samplewhen no such antibodies are present. Such shortcomings may posedifficulties in accurate detection of the presence of anti-ZIKVantibodies in a sample. According to the present disclosure, smallvolumes of sample may be used to generate high-probability detection ofanti ZIKV-antibodies in a sample.

In one aspect, disclosed is a method for detecting the presence ofanti-Zika virus antibodies, such as in a sample from a subject. Suchdetection may indicate current or prior infection with ZIKV. Inparticular, the presence of antibodies to the NS5 protein of ZIKV may bedetected. The presence of different antibody types, including IgG, IgM,or both, may be accomplished in accordance with the present disclosure.As would be understood by skilled artisans, the presence of differentantibody types following a subject's exposure to ZIKV or otherflaviviruses follows different time frames, with IgM antibodies toparticular antigens being produced sooner after infection than IgGantibodies to said antigens. In various embodiments, detection of one,the other, both, and/or other types of antibodies to ZIKV-derivedantigens may provide an indication of a time frame of a subject'sexposure to ZIKV relative to when a sample was obtained from thesubject. For example, presence of IgM anti-ZIKV antibodies may signifymore recent exposure while presence of IgG anti-ZIKV antibodies maysignify less recent exposure.

In another aspect, presence of antibodies to ZIKV NS5 may be performed.ZIKV NS5 protein is encoded for by nucleotides 7668-10376 of the ZIKVgenome (GenBank number KU955593.1; Shan et al., 2016, Cell Host &Microbe 19:891-900). This sequence, and the peptide product thereof,ZIKV NS5 peptide, differ from the genetic and peptide sequences of NS5for other viruses, including flaviviruses. For example, NS5 of DENV1-4differ from ZIKV NS5. As disclosed herein, affinity of antibodies toZIKV NS5 produced by a subject subsequent to exposure to ZIKV may differfrom the affinity of said antibodies to NS5 protein of other viruses,such as DENV or other flaviviruses. The detection of the presence of ananti-ZIKV NS5 antibody in a subject on the basis of whether saidantibody binds to ZIKV NS5 may therefore signify that the subject wasexposed to ZIKV as distinguished from having been exposed to DENV.Specifically, identifying antibodies in a subject that bind ZIKV NS5 inaccordance with the present disclosure indicates that the subject wasexposed to ZIKV and may be distinguished from an indication that saidsubject was exposed to DENV, or other viruses such as otherflaviviruses.

In another aspect, presence of antibodies to ZIKV NS1 may be detected.ZIKV NS1 protein is encoded for by nucleotides 2490-3545 of the ZIKVgenome (see, e.g., GenBank accession number KU955593.1; Shan et al.,2016, Cell Host & Microbe 19:891-900). This sequence, and the peptideproduct thereof, ZIKV NS1 peptide, differ from the genetic and peptidesequences of NS1 for other viruses, including flaviviruses. For example,NS1 of DENV1-4 differ from ZIKV NS1. Gene and peptide sequence of NS1for each of DENV1-4 also differ from each other. The complete genomes ofDENV1, DENV2, DENV3, and DENV4 are known (see, e.g., GenBank accessionnumbers NC_001477.1, NC_001474.2, NC_001475.2, and NC_002640.1,respectively). As disclosed herein, affinity of antibodies to ZIKV NS1produced by a subject subsequent to exposure to ZIKV differs from theaffinity of said antibodies to NS1 protein of other viruses, such asDENV1-4 or other flaviviruses. Nucleotide sequences for DENV1-4 NS1, andthe amino acid sequences of such proteins, is known (see, e.g., GenBankaccession numbers NP_722461.1, NP_739584.2, YP_001531169.2, andNP_740318.1 for DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1,respectively).

In another aspect, presence of antibodies to a flavivirus envelopeprotein may be detected. ZIKV envelope peptide amino acid sequence isamino acids 291-792 of ZIKV polyprotein (GenBank accession numberAAV34151). Envelope protein of other strains of ZIKV may also be used,as may Envelope proteins from other flaviviruses, such as DENV1 envelopeprotein, DENV2 envelope protein, DENV3 envelope protein, or DENV1envelope protein, given the highly conserved amino acid sequences ofenvelope protein among these viruses and strains thereof.

As further disclosed herein, affinity of antibodies to DENV1 NS1, DENV2NS1, DENV3 NS1, or DENV4 NS1 produced by a subject subsequent toexposure to DENV1-4, respectively, may differ from the affinity of saidantibodies to NS1 protein of other viruses, such as DENV serotypes towhich the subject was not exposed, or other flaviviruses. The detectionof the presence of an anti-ZIKV NS1 antibody in a subject on the basisof whether said antibody binds to ZIKV NS1 may therefore signify thatthe subject was exposed to ZIKV as distinguished from having beenexposed to DENV1-4. Furthermore, the detection of the presence of ananti-DENV1 NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 antibody in asubject on the basis of whether said antibody binds to DENV1 NS1, -DENV2NS1, -DENV3 NS1, or -DENV4 NS1, respectively, may therefore signify thatthe subject was exposed to DENV1, DENV2, DENV3, or DENV4, respectively,as distinguished from having been exposed to another serotype of DENV,ZIKV, or to another flavivirus. Specifically, as disclosed herein,identifying antibodies in a subject that bind ZIKV NS1 in accordancewith the present disclosure indicates that the subject was exposed toZIKV and may be distinguished from an indication that said subject wasexposed to DENV, or other viruses such as other flaviviruses. And, asdisclosed herein, identifying antibodies in a subject that bind to DENV1NS1, -DENV2 NS1, -DENV3 NS1, or -DENV4 NS1 in accordance with thepresent disclosure indicates that the subject was exposed to DENV1,DENV2, DENV3, or DENV4, respectively, and may be distinguished from anindication that said subject was exposed to another serotype of DENV, orto ZIKV or another flavivirus.

Antibodies produced in response to ZIKV exposure that have affinity forZIKV Envelope protein (E) may also have affinity for E protein of otherflaviviruses, such as DENV or other types of flaviviruses. Similaritiesof the peptide sequences and/or conformations of E protein of differentflaviviruses may result in the production of antibodies that arecross-reactive to various flaviviruses. If a subject has been exposed toZIKV, the subject may produce antibodies that have affinity not only forZIKV E protein but also for E protein of other flaviviruses, includingDENV. Likewise, a subject who has been exposed to DENV1-4 may produceantibodies with affinity for E protein of other flaviviruses. Detectionin a subject of an antibody with affinity for ZIKV E protein may signifythat the subject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, oranother flavivirus. And, detection in a subject of an antibody withaffinity for DENV1 E, DENV2 E, DENV3 E, or DENV4 E may signify that thesubject was exposed to ZIKV, DENV1, DENV2, DENV3, DENV4, or anotherflavivirus.

As further disclosed herein, in one embodiment, detection of multipledifferent antibodies may be performed on a given sample or samples froma given subject or subjects. In some embodiments, a sample from asubject may be tested for the presence of an antibody with affinity forZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, DENV4 NS1,or any combination of two or more of the foregoing. In one non-limitingexample, a sample from a subject may be tested for the presence ofantibodies to ZIKV NS5, ZIKV NS1, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3NS1, and DENV4 NS1. In another non-limiting example, a sample from asubject may be tested for the presence of antibodies to ZIKV NS1, ZIKVE, DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1. The presence of IgGand IgM antibodies to the foregoing antigens may be tested. If a subjecthas been exposed to ZIKV but not to other flaviviruses, IgG antibodies,IgM antibodies, or both, to ZIKV NS1, ZIKV NS5, and ZIKV E may bedetected. If the subject has been exposed to DENV1-4, antibodies toDENV1-4 NS1 may be detected, as well as to ZIKV E owing to thesimilarities between ZIKV E and DENV E and cross-reactivity ofantibodies thereto. If a subject has been exposed to both ZIKV and DENV,antibodies to ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3NS1, and DENV4 NS1 may all be detected in a sample or samples from saidsubject.

In a particular embodiment, presence or absence of anti-ZIKV NS5antibodies may be detected in a sample from a subject. As disclosedherein, high selectivity of anti-ZIKV NS5 antibodies for ZIKV NS5relative to NS5 or other proteins from other flaviviruses, such as DENV,corresponds to low cross-reactivity of anti-ZIKV NS5 antibodies forother flaviviruses. Similarly, as disclosed herein, low cross-reactivityof antibodies produced in response to exposure to flaviviruses otherthan ZIKV, such as DENV, to ZIKV NS5 results from selectivity ofantibodies produced in response to expose to said other viruses relativeto ZIKV NS5. Detection of the presence of an anti-ZIKV NS5 antibody in asample from a subject as disclosed herein indicates that the subject hasbeen exposed to ZIKV. The absence of anti-ZIKV NS5 antibodies in asample from a subject may signify that the subject was not exposed toNS5, even if the subject was exposed to another virus, such as anotherflavivirus such as DENV.

In another particular embodiment, presence or absence of anti-ZIKV NS1antibodies may be detected in a sample from a subject. As disclosedherein, high selectivity of anti-ZIKV NS1 antibodies for ZIKV NS1relative to NS1 or other proteins from other flaviviruses, such as DENV,corresponds to low cross-reactivity of anti-ZIKV NS1 antibodies forother flaviviruses. Similarly, as disclosed herein, low cross-reactivityof antibodies produced in response to exposure to flaviviruses otherthan ZIKV, such as DENV, to ZIKV NS1 results from selectivity ofantibodies produced in response to expose to said other viruses relativeto ZIKV NS1. Detection of the presence of an anti-ZIKV NS1 antibody in asample from a subject as disclosed herein indicates that the subject hasbeen exposed to ZIKV. The absence of anti-ZIKV NS1 antibodies in asample from a subject may signify that the subject was not exposed toNS1, even if the subject was exposed to another virus, such as anotherflavivirus such as DENV.

In another particular embodiment, presence or absence of anti-ZIKVenvelope protein antibodies may be detected in a sample from a subject.In particular examples, presence or absence of anti-ZIKV NS1, anti-ZIKVNS5, anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1,anti-DENV3 NS1, and anti-DENV4 NS1 may all be detected in a sample froma subject. In another example, presence or absence of anti-ZIKV NS1,anti-ZIKV envelope protein, anti-DENV1 NS1, anti-DENV2 NS1, anti-DENV3NS1, and anti-DENV4 NS1 may all be detected in a sample from a subject.

In some embodiments, for each and every of the anti-flaviviral proteinantibodies disclosed herein, an assay for detection thereof may generatesome level of signal indicating presence of reactive antibody in asample from a subject known not to have been exposed to a or theflaviviruses of interest. Such background signal is known by thoseskilled in this field to occur and is attributable to some baselinereactivity of sample constituents with a test antigen used as disclosedherein for identifying presence of an anti-flaviviral antibody.Conventionally, an average level of such background may be determinedfor a given assay against which detection results for a tested subjectmay be compared to determine whether a given antibody is present. Forexample, a number of subjects known or believed not to have been exposedto ZIKV or DENV may be tested for the presence of anti-ZIKV andanti-DENV protein antibodies as disclosed herein and an average level ofbackground reactivity of assays as disclosed herein may be determinedfor a given assay.

For subsequent testing of a subject who may or may not express anti-ZIKVor anti-DENV protein antibodies, a sample taken from such subject may betested in an assay as disclosed herein and results compared to anaverage from tests of subjects known or believed not to have beenexposed to such flaviviruses. In some instances, a level of detectionsignal some degree or amount above an average background level ofexpression may be selected as minimum level qualifying for evincingpresence of a given anti-flaviviral protein antibody in a sample from asubject. If a subject is tested for the presence of antibodies againstone or more of the foregoing flaviviral proteins and the level ofdetection signifying presence of any one or more of such antibodiesfalls below, or above, such cutoff value, then such antibodies may bedetermined not to have been detected, or to have been detected,respectively, in such sample. In some examples, a cutoff value may be acertain degree above an average level of background signal detected insubjects known or believed not to have been exposed to the flavivirusesin question. For example, a cutoff may be a number of standarddeviations above such mean background. A cutoff value may be one, two,or three standard deviations above mean background expression, meaningthat antibody to a given flaviviral protein is determined to have beendetected for a sample when an assay yields a given level of signalsignifying such presence. Cutoff levels may be determined on ananti-flaviviral protein antibody by anti-flaviviral protein antibodybasis, as well as on an assay-by-assay basis.

In some embodiments, if a test detects presence of anti-flaviviralproteins, a test may be repeated at a later date and comparingindications that such antibodies were detected at different testsperformed. For example, presence or absence of an IgG antibody, or anIgM antibody, or both types of antibodies, to one or more of theflaviviral proteins disclosed herein, may be detected. For example, IgMantibodies to ZIKV envelope and ZIKV NS1, may be detected. Such resultsmay indicate current or recent infection with ZIKV, such as inapproximately the past 21 days, as would detection of IgM antibodies toZIKV NS5. In another example, IgG antibodies to ZIKV envelope and ZIKVNS1, may be detected. Such results may indicate past infection withZIKV, such as more than 21 days ago, as would detection of IgGantibodies to ZIKV NS5. In still another example, IgM and IgG antibodiesto ZIKV envelope and ZIKV NS1, may be detected. Such results mayindicate current or recent infection with ZIKV, such as in approximatelythe past 21 days, as well as past infection, such as more thanapproximately 21 days ago, as would detection of IgM and IgG antibodiesto ZIKV NS5.

In still further embodiments, an avidity assay may be performed todetermine whether an anti-flaviviral protein antibody, such as anti-ZIKVenvelope protein, anti-ZIKV-NS1, anti-ZIKV NS5, anti-DENV1 NS1,anti-DENV2 NS2, anti-DENV3 NS1, or anti-DENV4 NS1 antibody, or anycombination of two or more of the foregoing antibodies, are present andif so, whether they bind to ZIKV envelope protein, ZIKV-NS1, ZIKV NS5,DENV1 NS1, DENV2 NS2, DENV3 NS1, or DENV4 NS1, respectively, with highor low avidity. Detection of high-avidity-binding antibodies that bindto a flaviviral protein with relatively higher avidity (e.g., anti-ZIKVenvelope/ZIKV envelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5,anti-DENV1 NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3NS1, or anti-DENV4 NS1/DENV4 binding) may indicate a subject wasinfected with a given flavivirus as disclosed herein longer ago.Detection of low-avidity-binding antibodies that bind to a flaviviralprotein with relatively low avidity (e.g., anti-ZIKV envelope/ZIKVenvelope, anti-ZIKV NS1/ZIKV/NS1, anti-ZIKV NS5/ZIKV NS5, anti-DENV1NS1/DENV1 NS1, anti-DENV2 NS1/DENV2 NSI, anti-DENV3 NS1/DENV3 NS1, oranti-DENV4 NS1/DENV4 binding) may indicate a subject was infected with agiven flavivirus more recently, or is currently infected with a givenflavivirus. High avidity binding is binding that persists or is stilldetectable after incubation of a solution of a flaviviral protein orcomposition such as a polyplex microsphere bound to such antigen asdisclosed herein with urea. Urea is known to disrupt low-avidityantigen-antibody association. When a sample is incubated with urea(e.g., 6 M, 8 M, or 10 M urea for 10 minutes) yet presence ofanti-flaviviral antibody as disclosed herein is still detected, thenhigh-avidity binding antibodies that bind to a given flaviviral proteinhave been detected. Detection of absence of antibody after ureaincubation indicates absence of high-avidity antibodies. Such aviditytesting may be performed for any one, all or any combination of assaysfor presence of anti-flaviviral protein antibodies.

In some embodiments, presence or absence of a given anti-flaviviralprotein antibody or antibodies may indicate that a subject from which atested sample was taken was previously exposed to or infected with orwhose body presently or formerly contained a given flavivirus. Forexample, if anti-ZIKV envelope protein antibodies are detected in asample, a subject may be determined to have had or presently have aflavivirus. Given cross-reactivity of an anti-ZIKV envelope proteinantibody with envelope proteins of other flaviviruses, such as DENV1,DENV2, DENV3, or DENV4, for example, apparent detection of an anti-ZIKVenvelope antibody may signify that the subject has had or has ZIKV,DENV1, DENV2, DENV3, or DENV4. By contrast, as disclosed herein, givenselectivity of antibodies to ZIKV NS1 to binding to ZIKV NS1 relative tobinding to other flaviviral proteins, such as DENV1 NS1, DENV2 NS1,DENV3 NS1, or DENV4 NS1, which is low, detection of anti-ZIKV NS1antibodies as disclosed herein signifies that a subject has or has hadZIKV in particular. Similarly, as disclosed herein, given selectivity ofantibodies to ZIKV NS5 to binding to ZIKV NS5 relative to binding toother flaviviral proteins, such as DENV1 NS5, DENV2 NS5, DENV3 NS5, orDENV4 NS5, which is low, detection of anti-ZIKV NS5 antibodies asdisclosed herein signifies that a subject has or has had ZIKV.

As further disclosed herein, detection of presence multiple differentanti-flaviviral protein antibodies combined with detection of absence ofe multiple different anti-flaviviral protein antibodies in combinationpermits possible discriminative determination of presence, or priorpresence, of ZIKV, DENV, or both, in a subject. For example, a subjectmay have had or may have ZIKV but not DENV of any serotype, in whichcase presence of anti-ZIKV NS1, anti-ZIKV-NS5, and anti-ZIKV envelopeprotein antibodies may be determined to be present in a sample from suchsubject, whereas anti-DENV NS1, anti-DENV2 NS1, anti-DENV3 NS1, andanti-DENV4 NS1 antibodies may be determined not to be present in samplesfrom such patients. In another example, a subject may have had or mayhave a flavivirus of a DENV serotype but not ZIKV, in which case absenceof anti-ZIKV NS1 and anti-ZIKV-NS5 antibodies may be detected, andpresence of anti-ZIKV envelope protein antibodies, and anti-DENV NS1,anti-DENV2 NS1, anti-DENV3 NS1, and anti-DENV4 NS1 antibodies (dependingon which DENV serotype the subject had or has), may be determined to bepresent in samples from such patients.

The presence of an antibody to a given antigen as disclosed herein maybe performed by any of a number of different known methods. In oneembodiment, a microsphere immunoassay (MIA) may be used for thedetection of an antibody to a given antigen. As would be appreciated byskilled artisans, rapid detection of multiple assays can be performed onlow volumes of specimen taken from a subject. In some embodiments,presence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, andNS1 proteins of any of DENV1-4 may be identified in a sample from asubject. In accordance with above-identified differential responsivenessto such testing on the basis of prior viral exposure, differentcombinations of positive results for particular antibodies may indicateexposure of the subject to ZIKV, DENV1-4, or another flavivirus.

Several options for MIA are available. In some embodiments, flaviviralantigens may be covalently coupled to microbeads and contacted to asample from a subject, whereupon antibodies to the given antigen presentin the sample bind to the bead via the antigen. Subsequentidentification of antibody bound to the bead indicates that antibodiesto the antigen were present in the sample. In other embodiments, antigenmay be coupled to microbeads that have an anti-antigen antibody, orother molecule designed to bind to the antigen such as an affimer oraptamer or other designer ligand, fused thereto via binding to saidantibody. Subsequent contacting of the antigen-coupled microbeads tosample from a subject may then be performed as described.

Different sources of viral antigen may be used in accordance with thepresent disclosure. For example, recombinant peptides whose sequencecorrespond to the sequence of viral peptide of interest (i.e., ZIKV NS1,ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be synthesizedby well-known methods and purified. Recombinant peptides may differ insequence from native of wild-type flaviviral protein, provided itretains structural, sequential, or conformational properties sufficientto permit recognition by antibodies generated in a subject subsequent toexposure to the corresponding flavivirus. flaviviral protein from whichthe to which the recombinant Different assays may detect whetherantibodies to flaviviral antigen in a sample are IgG antibodies or IgMantibodies, by known methods (e.g., IgG- or IgM-specific methods ofdetection of anti-flaviviral antigen antibody bound to microbeads,depleting IgG or IgM antibodies from a sample before contacting thesample with antigen-coupled beads, or selecting out IgG or IgMantibodies from a sample and contacting the selected-out antibodies withantigen-coupled microbeads.

In another embodiment, ELISA could be used to detect the presence ofantibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, and NS1proteins of any of DENV1-4 in a sample. For example, anti-IgM antibodiescan be coated onto a plate, such as a 96-well or other multi-well plate,and contacted with sample from a subject. IgM antibodies from the samplewould become bound to the anti-IgM-coated plate. Following well-knownprotocols, the IgM-bound plate could then be contacted with antiviralprotein of interest, such as ZIKV NS1, ZIKV NS5, ZIKV E, and NS1proteins of any of DENV1-4, followed by contact with a visualizableantibody to the viral protein. In this way, antiviral protein ofinterest would, via binding to any antibodies thereto present in thesample that had become bound to the anti-IgM antibody on the plate, bebound to plates that had been exposed to samples from subject that hadbeen exposed to the flavivirus. In turn, such samples would bevisualizable, via binding of visualizable anti-flaviviral protein, suchas through conjugation with antibodies, fluorophores, or otherwell-known visualizable or detectable markers. Upon processing to detectpresence of the visualizable marker, plates that had been contacted witha sample from a subject that had been exposed to the correspondingflavivirus can be identified and distinguished from those that have not.By performing MAC-ELISA on a sample for antibodies to several differentflaviviral proteins (for example, any combination of two or more of ZIKVNS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4), it can bedetermined whether the subject had been exposed to ZIKV, DENV, and/oranother flavivirus.

In another embodiment, a dot blot may be performed to detect thepresence of antibodies to one or more of ZIKV NS1, ZIKV NS5, ZIKV E, andNS1 proteins of any of DENV1-4. In dot blotting, a spot or dot ofantigen is applied to a membrane, such as nitrocellulose orpolyvinylidine fluoride (PVDF). The membrane may then be exposed to asample from a subject. If the subject has been exposed to a virus thatcontains the antigen applied to the membrane, such antibodies willcouple to a region of the membrane to which the antigen has beenapplied. The membrane can then be contacted by secondary antibodies(e.g., anti-IgM or anti-IgG) coupled to a visualizable marker (e.g.,radiolabel, fluorescent tag, magnetic tag, enzyme, etc.). Visualizablemarker is thereby coupled to any region of the membrane to which anantigen recognized by antibodies thereto present in the sample. Upondetection of said marker, presence of antibodies to said antigen in thesample may be determined. In some embodiments, a dot blot analysis maybe performed to detect the presence of antibodies to any one or more ofZIKV NS1, ZIKV NS5, ZIKV E, and NS1 proteins of any of DENV1-4, and anycombination of two or more of the foregoing. As described above,different permutations of positive and negative results for differentanti-flaviviral antigen antibodies suggests that the subject from whichthe sample was taken was exposed to different flavivirus(es).

In yet another embodiment, immunochromatography may be used to identifythe presence of one or more flaviviral antibody in a subject. As onenonlimiting example, and immunochromatographic strip test (ICST) may beused. In such assays, a small strip comprising capillary beds may beexposed to a sample from a subject. Embedded in the strip may be a viralantigen to which antibodies are to be detected. When an aqueous sampleis drawn into contact with such antigens following contact with thecapillary structure of the strip, the antigens are bound to anyantibodies thereto that are present in the sample. The antigen may carryvisualizable markers, such as colored latex, gold nanoparticles, orfluorescent or magnetic particles which can be visualized withappropriate readers. Further along the strip, a capture portioncontaining antibodies to the antigen immobilized to the strip bed may bepresent, such as in a concentrated region, stripe, or area. As samplecontinues to migrate along the capillary bed, it encounters theimmobilized antibodies of the capture portion. If antibody to theparticular antigen were present in the sample, their migration acrossthe capillary bed would have carried bound, visualizable antigen withthem, which, in turn, would be immobilized on the capture portion owingto the anti-antigen antibodies immobilized therein. As more antibodyaccumulates that the capture portion, the visualizable markers becomedetectable and, when detected, signify the presence of antibody to theantigen of interest.

In some examples, multiple ICST strips, each designed to permit thedetection of antibodies to a different flaviviral antigen (e.g., ZIKVNS1, ZIKV NS5, ZIKV E, or NS1 proteins of any of DENV1-4) may be used totest the presence of antibodies to more than one such antigen in asample. As described above, different permutations of positive andnegative results for different anti-flaviviral antigen antibodiessuggests that the subject from which the sample was taken was exposed todifferent flavivirus(es).

As would be appreciated by skilled artisans, modifications of theforegoing methods may be performed in keeping the with presentdisclosure. Other methods where ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1,DENV2 NS1, DENV3 NS1, DENV4 NS1, or combinations of any two or more ofthe foregoing, are used to bind antibodies present in a sample, followedby methods used to visualize, detect, quantify, bind, identify, orotherwise record the presence and/or quantity thereof in the sample maybe performed, all of which are explicitly intended as embodiments of thepresent disclosure, of which the foregoing examples are but non-limitingrepresentations of the method disclosed herein. Kits containing reagentsused in performing any such method, including reagents, antigens,labeling reagents or labeled antigens or antibodies, microspheres,binding membranes such as nitrocellulose, PVDF, or the like, lateralflow strips, with or without reagents needed for performing thedisclosed method included therein, any tubes, vials, needles, or othertools needed for obtaining and/or storing samples, etc., are explicitlyincluded in and considered embodiments of an invention disclosed herein.

As would be understood by skilled artisans, antigens used in any of theforegoing methods may be modified in accordance with standard molecularbiology techniques. For example, antigens may be produced withidentifiable tags appended thereto to facilitate isolation,identification, concentration, collection, application, or other usesthereof. As one non-limiting example, antigens with histidine tags maybe produced to facilitate separation of recombinant antigens from otherconstituents employed during synthesis thereof, and subsequentconcentration of said antigens in a usable form, solvent, diluent, orconcentration. Although histidine tags are mentioned here as onenon-limiting example, skilled artisans would appreciate that any of awide variety of such tools could be adapted to and employed with theabove-disclosed methods and compositions, without departing from, andhereby explicitly recounted as included within, the method as disclosedherein.

Although non-limiting examples discussed herein include usingfull-length recombinant flaviviral peptides as antigens, skilledartisans would appreciate that full-length peptides would notnecessarily be required for use in all embodiments of the method andcompositions disclosed herein. For example, a specific or portions lessthan full-length viral proteins may be used rather than full-lengthprotein. A portion less than full-length protein to which antibodiesproduced in response to a subject's exposure to a given flavivirus willbind may be used rather than full-length protein, for example. In someembodiments, a portion less than full-length flaviviral protein to whichantibodies produced in response to a subject's exposure to a givenflavivirus, but not another flavivirus, will bind may be used. In otherexamples, portions less than full length protein that bind to antibodiesproduced in response to either of two or more different flaviviruses maybe used.

In yet other embodiments, an antigen with less than total homology to aflaviviral protein, such as ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2NS1, DENV3 NS1, or DENV4 NS1 may be used, as may a combination of anytwo or more of the foregoing. For example, a recombinant flaviviralprotein with an amino acid sequence that corresponds to some but not allof the amino acids in the flaviviral protein to which it corresponds maybe used. In some examples, a recombinant flaviviral antigen with 75% ormore homology to a corresponding naturally occurring flaviviral proteinmay be used. In some examples, a flaviviral antigen with between 75%-80%homology, 80%-85% homology, 85%-90% homology, 90%-95% homology, 95%-100%homology, 90%-91% homology, 91%-92% homology, 92%-93% homology, 93%-94%homology, 94%-95% homology, 95%-96% homology, 96%-97% homology, 97%-98%homology, or 99%-100% homology may be used. Several strains of ZIKV havebeen identified with high levels of homology between their sequences ofZIKV envelope protein, between their sequences of ZIKV NS1, and betweentheir sequences of ZIKV NS5. ZIKV protein sequences homologous to suchknown strains may be used, within homology ranges as disclosed herein,in accordance with the present disclosure. A recombinant flaviviralantigen with less than 100% homology to a corresponding flaviviralprotein to which antibodies produced in response to a subject's exposureto a given flavivirus will bind may be used rather than a recombinantprotein with 100% homology. In some embodiments, a recombinantflaviviral protein of less than 100% homology to flaviviral protein towhich antibodies produced in response to a subject's exposure to a givenflavivirus, but not another flavivirus, will bind may be used. In otherexamples, a recombinant flaviviral protein of less than 100% homology toflaviviral protein to which antibodies produced in response to asubject's exposure to two or more given flaviviruses may be used.

In other embodiments, an antigen with an amino acid sequence thatcorresponds to only a portion of a full-length flaviviral protein, suchas ZIKV NS1, ZIKV NS5, ZIKV E, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4NS1 may be used, as may a combination of any two or more of theforegoing. For example, a truncated portion of ZIKV NS1, ZIKV NS5, ZIKVE, DENV1 NS1, DENV2 NS1, DENV3 NS1, or DENV4 NS1, which is recognized byantibodies generated in response to flaviviral infection, may be used.In some embodiments, an antigen may include amino acids not present innaturally occurring flaviviral proteins. For example, as would beunderstood by skilled artisans, for purposes of antigen synthesis andpurification, an antigen may contain an amino acid tag, such as on itsC-terminus or its N-terminus, without interfering with the bindingthereto of an antibody produced by an individual as a result offlaviviral infection. Many possible such tags ae well-known, including apoly-histidine tag, a myc-tag, a FLAG-tag, an HA-tag, or many others,all of which could be included as part of an antigen and be within thescope of methods and kits as disclosed herein.

A sample may be a biological sample, such as a bodily fluid, blood,serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissuehomogenate, cellular extract, or spinal fluid, containinganti-flavivirus antigen antibodies, such as IgG anti-NS5, IgM anti-NS5,IgG anti-E, or IgM anti-E. A subject may be any vertebrate, such as ahorse, pig, cow, dog, cat, bat, primate, including human, goat, sheep,deer, rabbit, mouse, rat, chicken or other avian species, or otheranimal.

EXAMPLES

The following examples are presented to further describe techniques inaccordance with the method disclosed herein, but should not be read aslimiting, because variations still within the scope of embodiments ofthe present invention will be apparent to those skilled in the art.

Reagents. Wash buffer and phosphate buffered saline pH 7.4, 0.05% sodiumazide (PBS-TN) were purchased from Sigma (Sigma Aldrich, St. Louis,Mo.). Chemicals, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), weresupplied by Pierce Chemicals (Pierce, Rockford, Ill.). Microspheres,calibration microspheres, and sheath fluid were obtained from LuminexCorporation (Luminex Corp., Austin, Tex.).

Serum samples. Studies were performed on serum from de-identifiedclinical specimens submitted to New York State Department of Health forZIKV IgM-capture ELISA and Arbovirus MIA testing.

Positive and negative serum controls. ZIKV positive control sera weredefined as positive titer from a Plaque Reduction Neutralization Test of90% inhibition (PRNT₉₀) against ZIKV, but negative PRNT₉₀ titer againstDENV. Similarly, DENV positive control sera were defined as positivePRNT₉₀ titer against DENV, but negative PRNT₉₀ titer against ZIKV.Negative control sera were defined as no ZIKV PRNT₉₀ titer as well asnegative Arbovirus MIA result using WNV E protein as the diagnosticantigen (Wong et al., 2003). PBN (consisting of PBS, 1% BSA, 0.05%Sodium Azide, pH 7.4) was used as a blank control.

Expression and purification of recombinant ZIKA NS5 protein. The cDNAfragment encoding the full-length NS5 of ZIKV was amplified from aninfectious clone pFLZIKV (Shan et al., 2016b), fused with a C-terminal(His)6-tag, and cloned into vector pNIC28-Bsa4 (GenBank accessionEF198106), resulting in plasmid construct pNIC28-ZIKA-NS5. ZIKA NS5protein was expressed in E. coli Rosetta 2 pLysS E. coli (Stratagene)and purified using a method as previously described (Zhao et al., 2015)with some modifications. Briefly, transformed E. coli cells was inducedby 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the celldensity reached OD600 of 0.6-0.8. After incubation at 18° C. for 16 h,the cells were harvested, re-suspended in buffer A (20 mM Tris-HCl, pH8.5, 550 mM NaCl, 10% glycerol, 5 mM β-mercaptoethanol, 10 mM imidazole,and 0.5×EDTA-free protease inhibitor cocktail) by sonication. The lysatewas clarified by centrifugation at 40,000 g for 30 min at 4° C. Theresulting supernatant was loaded onto a HisTrap Fast Flow column (GEHealthcare). The protein was eluted using a linear gradient of imidazoleconcentration from 40 to 500 mM. The fractions containing ZIKANS5-(His)6 protein were pooled, concentrated, and further purified bygel filtration using a HiLoad Superdex 200 16/60 column (GE Healthcare)in buffer B (20 mM Na-Hepes, pH 8.2, 500 mM NaCl, 10% glycerol, and 5 mMDTT). The peak fractions containing ZIKA NS5-(His)6 protein were pooledand concentrated to approximately 1-2 mg/ml before storage at −80° C.

Recombinant ZIKV E, NS1, and DENV NS1 proteins. Recombinant ZIKV E, NS1,and DENV-1 to DENV-4 NS1 proteins were purchased from Meridian (MeridianLife Science, Inc., Memphis, Tenn.). All Meridian recombinant proteinswere produced in insect cells and purified by affinity chromatographymethod. Purified proteins were analyzed by 12.5% sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (PAGE) and stored in PBS pH7.4.

Conjugation of protein antigens to microsphere Luminex beads.Recombinant proteins were covalently coupled to Luminex MicroPlexMicrospheres carboxylated polystyrene microparticles following apreviously reported protocol (Wong et al., 2003). Briefly, 50 μg ofpurified protein was used to couple to the surface of 6.25×106microspheres in a two-step carbodiimide process. (i) Activation ofmicrospheres. Microspheres were activated with 10 l ofN-hydroxysuccinimide (sulfo-NHS) (50 mg/ml) followed by 10 l of1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCl (50 mg/ml).Microspheres were then incubated for 20 min at room temperature withgentle vortexing at 10-min intervals. (ii) Coupling of recombinantproteins. Each recombinant protein was added to the activatedmicrospheres with distinct fluorescence. Protein-microsphere mixtureswere incubated for 3 h in the dark on a LabTech tube rotator(Barstead/Thermolyne, Dubuque, Iowa). The microspheres were then washedtwice by centrifugation and resuspended in 1.0 ml PBS-TN [phosphatebuffered saline pH 7.4, 0.05% sodium azide, 1% bovine serum albumin(BSA)]. The protein coupled microsphere were then stored at 4° C.

Multiplex reagent preparation and microsphere immunofluorescence assay(MIA) procedure. All reagent dilutions and assays were carried out inPBS-TN (phosphate buffered saline pH 7.4, 0.05% sodium azide, 1% BSA)(Sigma Aldrich, St. Louis, Mo.). The bead mixture consisted of sevenbeads, each coupled with a different flavivirus recombinant protein.Three bead sets contained ZIKV E (Meridian), NS1 (Meridian), and NS5(described above). Four other bead sets contained NS1 proteins fromDENV-1 to -4. The bead sets were stored at 4° C. in the dark and diluted1:100 directly in PBS-TN before use. Biotin conjugated goat anti-humanIgG/A/M affinity purified secondary antibody (Life Technologies, GrandIsland, N.Y.) was diluted 1:8000 in PBS-TN directly before use.Streptavidin-R-phycoerythrin (1 mg/ml SA-PE, Life Technologies, GrandIsland, N.Y.) was diluted 1:100 in PBS-TN before use. All serum sampleswere stored at −80° C. Samples were thawed and diluted 1:100 in PBS-TNright before use. Diluted samples were used within 1 h post dilution.

A 96-well MultiScreenHTS BV 1.2 μm Filter Plate (Millipore Billerica,Mass.) was wetted with 100 μl PBS-TN and washed once with washing buffer(PBS, 0.05% Tween 20, pH 7.4; Sigma Aldrich, St. Louis, Mo.). Samples(50 μl) were dispensed in each well to which 50 μl bead mixture wasadded. The plate was incubated in the dark on a shaker for 30 min andwashed three times with 190 μl washing buffer. After addition of 50 μlconjugate antibody, samples were incubated in the dark on a shaker for30 min and washed three times with 190 μl wash buffer. After addingdetection reagent SA-PE (50 μl), samples were incubated in the dark on ashaker for 30 min, washed twice with 190 μl wash buffer, and transferredto a flat bottom 96-well plate (Corning Incorporated, Kennebunk, Me.).Analysis was performed using a Luminex 100 Analyzer configured to count100 beads per bead class and a 100 μl sample size.

For avidity assays, urea at a final concentration of 8 M was added to asample during incubation of the sample with microspheres bound to agiven antigen and urea incubation was for 10 min. Anothermicrosphere-sample was incubated in the absence of urea in parallel.Median fluorescence intensity of each sample was then determined. AnAvidity Index was identified, which is a ratio of median fluorescenceintensity of urea-treated sample divided by median fluorescenceintensity of non-urea treated sample, times 100. An IgG Avidity Index ofless than or equal to 30% indicates infection occurred within the past65 days, or early acute infection. An IgG Avidity Index of from 31% to60% indicates infection occurred between 65 and 190 days ago, or lateacute infection. An avidity index of greater than or equal to 60%indicates infection occurred more than 190 days ago. In samples in whichanti-Zika NS1 or anti-Zika NS5 antibodies are detected in addition toanti-DENV antigen antibodies, avidity testing assists in determiningwhether one infection preceded the other, or whether one or the otherinfection is current or more acute than the other. For example, asubject may have a high Avidity Index for anti-DENV antigens and a lowAvidity Index for anti-ZIKV antigens, indicating recent or current ZIKVinfection and past DENV infection.

Results

Rationale of assay platform and antigen selection. The MIA platform forassay development was used because of (i) its multiplex capability tosimultaneously detect antibodies against a number of viral proteins,(ii) rapid assay turnaround time in <4 h, and (iii) low specimen volumerequirement (10 μl serum). This is in contrast with the well-establishedIgM-capture ELISA platform that requires >2 days of the assay turnaroundtime. For antigen selection, three recombinant ZIKV proteins wereemployed for the multiplex assay: E, NS1, and NS5. ZIKV E protein wasincluded to sensitively detect viral infection; however, due to thecross-reactive nature of E antibodies among flaviviruses, an E-positivesignal does not confirm ZIKV infection. ZIKV NS1 and NS5 proteins wereincluded to improve assay specificity through detection of virus-typespecific antibodies. In addition, recombinant DENV NS1 proteins fromeach of the four serotypes were included for testing virus-typespecificity. All antigens, except ZIKV NS5, were commercially purchased.ZIKV NS5 was cloned, expressed, and purified to >95% homogeneity. SeeFIG. 1. SDS-PAGE analysis of recombinant ZIKV NS5 protein. Full-lengthNS5 of ZIKV was expressed in an E. coli system, and purified throughaffinity followed by size-exclusion chromatography (see details inMaterials and Methods). The recombinant protein was analyzed on a 12%Mini-PROTEAN® TGX Stain-Free™ Protein Gel (Bio-rad).

Establishment of multiplex MIA. Seven recombinant antigens describedabove (ZIKV E, NS1, NS5, and DENV-1 to -4 NS1) were individuallyconjugated to microsphere beads, each with a distinct fluorescentsignature. A mixture of seven antigen-conjugated beads were reacted withpatient serum and quantified by anti-human immunoglobulins (reactivewith IgG, IgM, and IgA) with a red fluorescent phycoerythrin. Toestablish the cutoff level for each antigen, we assayed 20 presumedhuman sera from healthy individuals in the context of multiplex MIA. Theresults revealed cutoff values (defined as mean plus three timesstandard deviations) to be 1363, 284, 1905, 746, 549, 339, and 655 forZIKV E, NS1, NS5, and DENV-1, -2, -3, and -4 NS1, respectively. Thesecutoff values were used to determine positive (>cutoff) and negative(<cutoff) when diagnosing patient specimens.

Stratification of patient sera. A well-defined set of patient specimensmay be used to develop and verify the multiplex assay. A total of 154patient sera with known ZIKV and DENV PRNT results were selected forassay development. Based on the PRNT results, patient sera werecategorized into four distinct groups. Group I specimens (7 patients;Table 1) were both ZIKV- and DENV-negative, as defined by PRNTs<10 forboth viruses.

TABLE 1 Type I specimens with neither ZIKV nor DENV infection defined byPRNT* ZIKV IgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff 2 &Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 3 1363 284 1905746 549 339 655 <10 <10 0.2 1189 167 573 1200 935 100 450 <10 <10 0.51415 155 308 220 259 61 96 <10 <10 0.9 195 54 1128 87 51 230 175 <10 <101.9 366 91 1900 306 216 168 136 <10 <10 1.9 7742 4619 4267 127 105 113275 <10 <10 3.8 178 69 534 235 141 113 211 <10 <10 11.6 1001 53 211 418509 75 125 *The table is sorted with increasing value of IgM-captureELISA result.

Group II specimens (9 patients; Table 2) were ZIKV-negative andDENV-positive, as defined by ZIKV PRNT<10 and DENY PRNT>10.

TABLE 2 Type II specimens with DENV only infection defined by PRNT* ZIKVIgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNT PRNTELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff 2 & CutoffCutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 3 1363 284 1905 746 549339 655 <10 20 0.19  9131 234 170 5375 1579 3091 1442 <10 40 0.60  7145211 331 9101 2831 5415 3740 <10 80 1.5 11139 2514 249 9145 6658 1060914941 <10 160 1.5  9596 1806 177 7486 5198 8241 11867 <10 640 1.5  538146 227 5323 860 2235 1352 <10 80 1.6  8071 67 554 6862 510 1765 658 <1040 5.1  6017 51 1473   98 50 99 165 <10 160 5.8  7756 38 968 4259 179582 305 <10 40 77  8703 106 1009  8805 1511 3165 1558 *The table issorted with increasing value of IgM-capture ELISA result.

Group III specimens (43 patients; Table 3) were ZIKV-positive andDENV-negative, as defined by ZIKV PRNT>10 and DENV PRNT<10.

TABLE 3 Type III specimens with ZIKV only infection defined by PRNT *ZIKV IgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4 PRNTPRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff 2 &Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 3 1363 284 1905746 549 339 655 80 <10 0.6 9960 5207 2624 1346 1172 454 744 320 <10 0.9910692 6173 1676 409 531 660 631 2560 <10 1.8 16318 9407 10423 548 737802 492 320 <10 1.9 12377 6550 21510 391 644 927 422 40 <10 2.4 858 6171401 349 272 249 374 10 <10 2.75 2185 297 1777 795 729 189 285 80 <103.04 800 481 227 163 213 118 252 160 <10 3.80 9088 7010 3298 438 588 782459 320 <10 3.97 11272 4849 2129 1556 1268 250 838 320 <10 4 11358 49332179 1503 1212 212 735 320 <10 4.5 8592 5500 4362 311 455 452 220 2560<10 4.5 8592 5500 4362 311 455 452 220 160 <10 6.2 3896 3152 2524 574477 203 371 160 <10 7.88 5782 701 574 340 260 125 217 1280 <10 7.9 32335196 5865 231 263 315 246 1280 <10 7.9 4777 6771 8260 364 417 508 381320 <10 8.1 8842 4811 15599 214 349 561 276 320 <10 8.1 9768 5001 18463253 405 532 294 160 <10 8.29 457 540 930 464 258 204 168 1280 <10 10.9496 1010 3139 102 84 74 108 160 <10 13.5 12420 7611 1809 141 195 218 401160 <10 13.5 14292 8757 2491 180 243 265 548 160 <10 13.7 7432 6664 287690 159 337 149 40 <10 17 536 1554 2612 153 202 122 153 640 <10 17.706381 7713 18520 1095 1082 628 419 640 <10 20.1 2474 2312 2038 197 205 6782 640 <10 20.18 3703 1566 4269 75 69 314 54 160 <10 22 530 428 679 151116 110 119 160 <10 22 999 5442 3802 217 234 263 86 320 <10 25 7905 68145828 244 350 242 166 320 <10 25.5 8518 6914 6650 261 378 303 228 1280<10 27 8494 5334 3631 102 107 103 274 1280 <10 27.18 10443 6998 4530 157163 158 407 640 <10 28 3488 11213 4269 290 452 579 62 640 <10 28.8 87417471 3820 278 492 662 147 640 <10 29.9 6212 4915 5969 193 221 288 115640 <10 30 3998 3187 3867 133 141 158 73 1280 <10 32 11745 7674 4459 17449 242 250 1280 <10 32.3 13259 8154 4726 227 309 304 309 640 <10 42.19721 7456 658 143 276 230 139 2560 <10 47.2 16346 4928 3714 149 158 32060 320 <10 50.4 8250 7160 638 134 251 215 122 320 <10 65.6 3645 27851229 52 74 77 79 *The table is sorted with increasing value ofIgM-capture ELISA result.

Group IV specimens (95 patients; Table 4) were both ZIKV- andDENV-positive, as defined by PRNTs>10 for both viruses.

TABLE 4 Type IV specimens with ZIKV and/or DENV infection(s) defined byPRNT* ZIKV IgM- ZIKV DENV capture ZIKV ZIKV DENV-1 DENV-2 DENV-3 DENV-4PRNT PRNT ELISA ZIKV E NS1 NS5 NS1 NS1 NS1 NS1 Cutoff Cutoff Cutoff 2 &Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff Cutoff 10 10 3 1363 284 1905746 549 339 655 2560 320 0.3 12411 3002 6118 7992 5677 11340 10357 10 400.40 7446 285 790 7714 2875 5725 4420 320 2560 0.4 15916 4164 1195110070 8345 14047 13683 20 1280 0.41 18428 802 461 7969 5627 10631 958180 640 0.45 17887 2203 665 5073 3700 5778 9545 160 640 0.45 18357 2550796 5886 4181 6677 10700 20 640 0.45 17361 874 551 9206 6339 11251 1117110 80 0.49 16355 1950 570 10920 8275 15354 11740 1280 2560 0.53 188096682 650 16472 10777 18272 19435 10 160 0.53 8131 844 325 5142 3838 74636020 1280 2560 0.56 20863 6548 602 16500 10369 19309 19308 160 2560 0.6217472 3453 2503 12655 2806 11109 16787 160 2560 0.69 18220 3461 238413241 2871 12265 17857 2560 640 0.8 18354 8434 2396 382 426 556 309 6401280 1.09 16382 4065 3303 11394 7116 13885 7695 320 2560 1.3 17322 4085875 15507 11175 17637 16700 2560 320 1.41 19995 6926 8456 12524 1126817951 16740 640 1280 1.53 21501 7952 3037 16148 12733 19792 22445 320160 1.63 14009 6104 1374 11801 11004 17719 4475 320 2560 1.7 19788 86153494 16160 13589 19342 20432 160 2560 1.8 16928 3607 551 14462 1056117158 15002 1280 1280 1.81 17896 7868 2698 14294 11672 17853 21180 3202560 1.9 20374 8309 2755 16466 13975 19997 20689 640 2560 2.1 2102112154 15783 18993 14796 21073 22419 640 2560 2.16 21035 11444 1353618240 15675 21260 22412 5160 5120 2.3 18554 6707 553 12751 10464 1736220153 5120 1280 2.3 19606 11677 11105 14953 11886 18231 21051 5120 12802.32 18697 9335 7705 12257 9981 16633 17776 640 1280 2.45 20559 1093921381 14675 10696 18537 17415 2560 1280 2.5 19945 9641 13280 15589 1302219484 15561 2560 1280 2.52 18959 8035 11647 14771 12827 19260 13601 32040 2.57 9760 1309 386 5967 1610 2724 3719 160 40 2.59 10893 859 180 3100914 1643 6687 160 40 2.72 13602 1082 205 4179 1260 2191 8609 320 40 2.9910133 1442 417 6469 1696 3018 4257 320 5120 3.13 18591 7977 1050 119959617 15868 14587 160 320 3.53 19680 5587 1681 317 292 543 270 2560 25603.89 17268 4501 5464 13748 9869 17527 12169 5120 5120 3.99 21644 1085921825 12761 9859 17755 20283 160 320 4.02 16822 6103 2070 14021 1029817324 15517 640 1280 4.42 20593 5338 1739 12752 11158 18128 18042 12801280 4.43 19973 8594 11143 12763 11051 17739 16997 1280 320 4.79 1935510636 9796 12751 10778 18003 18073 640 1280 4.88 16101 4858 776 130938198 14431 8430 2560 1280 5.01 20780 12080 17177 16920 13760 20359 17012160 640 5.1 19275 6311 2744 14623 12984 19646 15016 160 640 5.34 162895747 2635 15009 11904 18826 16649 10 80 5.47 16794 1307 1328 389 200 346281 1280 640 6.05 16809 6987 3429 13666 12416 18579 5716 1280 80 6.522111 3626 1100 209 289 243 291 320 160 7.04 19585 2905 3390 10849 557910368 11556 320 160 7.04 20334 3419 4461 11917 6275 12136 12769 320 807.32 19940 2687 2942 10583 5368 9833 10471 1280 10 7.49 13338 9654 6618462 650 1220 700 1280 10 7.5 12624 9195 6717 341 478 986 514 2560 204808.3 20265 9171 2720 16781 12747 18712 20473 640 5120 8.5 20671 9098 707817100 14388 18608 22061 640 10240 9.04 21114 11671 11204 19358 1522321543 22106 2560 320 10.16 18279 9899 7213 4524 3165 6948 1278 160 64010.90 20183 7660 2078 2723 2628 5909 1384 320 640 10.99 14737 4336 1562611828 9458 15927 6841 320 2560 11.1 20977 10041 2254 17295 13477 1954910638 640 10240 11.4 20785 12169 11619 18884 15119 20667 21989 1280 256012 19968 8988 4259 17044 13238 19273 22230 1280 2560 12.54 20467 89013673 16470 13221 19341 22846 320 640 13 20546 6045 1818 15929 1097418017 3192 640 1280 14 18531 5202 1896 11359 10329 16770 16440 640 64014.64 20336 7675 1722 13716 12002 18849 20055 320 2560 15 21156 104633187 18463 14572 20246 22876 640 2560 16 20527 9862 5041 16141 1240819080 21991 640 2560 16.29 19340 9066 5185 15029 11748 17407 20104 256020480 17 22317 6665 7447 15855 10725 17403 5509 2560 20480 17.23 179995738 7214 13635 8930 14737 5748 160 1280 17.41 19848 8375 6839 1567913745 18998 22484 160 160 20.73 9391 6074 3340 356 679 566 309 10240 32022 17752 7270 4264 62 156 388 96 1280 1280 22.2 20185 8584 16360 1603012594 20100 10783 1280 20480 23 20703 10096 7859 18086 13614 18507 21267320 20 23.6 9924 8081 9837 113 203 469 231 1280 5120 23.69 18836 84984119 15799 12910 18325 20333 320 10 25 1061 3108 2294 88 89 83 894 640640 25.23 20066 9245 4529 17030 14304 20372 22431 2560 2560 26.13 211849524 2734 15615 13845 19053 22401 640 2560 27.9 19121 3787 3230 35223308 7426 3591 1280 10240 29.15 19711 10327 2437 16973 14262 18898 203411280 2560 29.9 21902 8590 3728 16006 13452 20836 22510 640 1280 30.5318080 8675 17545 16414 13525 18677 14452 2560 640 32 20207 6094 1156610231 7930 14480 11206 640 2560 33.4 20649 10126 6628 16806 13194 1875922293 320 160 35.88 4640 4268 2211 294 263 175 169 320 10 44.8 9837 72002590 284 253 308 194 2560 2560 46.1 19943 7114 7170 15766 11664 184548917 640 640 47.93 2828 5437 7229 334 1185 1407 299 1280 20 49.11 140636262 6839 359 291 193 250 1280 640 57 19062 6018 11756 12258 10914 1724017024 *The table is sorted with increasing value of IgM-capture ELISAresult.

It should be noted that, due to cross-neutralization of antibodies amongflaviviruses, group IV specimens could derive from patients who were (i)infected with both ZIKV and DENV, (ii) infected with ZIKV only but hadantibodies cross-reactive to DENV, or (iii) infected with DENV only butwith antibodies cross-reactive to ZIKV.

Multiplex MIA and IgM-capture ELISA diagnosis. Patient samples weresubjected to multiplex MIA and the well-established IgM-capture ELISA(Martin et al., 2000). Tables Si to S4 summarize the results for groupsI to IV specimens, respectively. Each specimen is presented with resultsfrom PRNT, IgM-capture ELISA, and multiplex MIA for individual antigens.It should be pointed out that, for IgM-capture ELISA, P/N<2 is definedas negative, P/N 2-3 as equivocal, and P/N>3 as positive. Table 5 inFIG. 2 summarizes the overall diagnostic results. For Table 5 in FIG. 2,results from Tables 1 to 4 are summarized for comparison of PRNT,IgM-capture ELISA, and multiplex MIA diagnosis. For each diagnosticparameter, the total number of samples that were diagnosed as “positive”(greater than cutoff line) or “negative” (less than cutoff line) isindicated, followed by its corresponding percentage of the total numberof specimens from that specific specimen group. Percentage (%)=(numberof positive or negative specimens/total number of specimen from thespecific specimen group)×100%.

For group I specimens (neither ZIKV nor DENV infection), both ZIKVIgM-capture ELISA and E MIA showed 71% negative; the MIA results fromZIKV NS1, ZIKV NS5, and combined DENV-1 to -4 NS1 showed 86% negative.For group II specimens (DENV infection only), ZIKV IgM-capture ELISAshowed 67% negative (i.e., 33% cross-reactivity with DENV); ZIKV E andcombined DENV-1 to -4 NS1 MIA showed 100% and 89% positive,respectively; in contrast, ZIKV NS1 and NS5 MIA showed 78% and 100%negative, respectively. For group III specimens (ZIKV infection only),ZIKV IgM-capture ELISA showed 86% positive; ZIKV E, NS1, and NS5 MIAshowed 84%, 100%, and 74% positive, respectively; whereas combinedDENV-1 to -4 NS1 MIA showed 63% negative (i.e., 37% cross-reactivitywith ZIKV). For group IV specimens (at least one infection from ZIKVand/or DENV), ZIKV IgM-capture ELISA, E, NS1, NS5, and combined DENV NS1showed 63%, 99%, 100%, 73%, and 96% positive, respectively. Theseresults enable the following analysis.

(I) Comparison of ZIKV IgM-capture ELISA and E MIA. Compared withIgM-capture ELISA, ZIKV E MIA alone showed equivalent accuracy whendiagnosing group III specimens, with 86% and 84% of the samples testedpositive from IgM-capture ELISA and E MIA, respectively. When diagnosinggroup IV specimens, the E MIA showed better sensitivity than IgM-captureELISA, with 63% and 99% of specimens tested positive, respectively. Twofactors may account for this improvement. (i) MIA measures IgG and IgAin addition to IgM, whereas IgM-capture ELISA does not capture IgG andIgA. (ii) The amount of IgM declines after the convalescent phase ofZIKV infection; therefore, specimens (taken long after convalescentphase) may have low levels of IgM and high levels of IgG, which is notdetected by the IgM-capture ELISA. Taken together, the results indicatethat E MIA alone has equivalent or better sensitivity than IgM-captureELISA.

(II) Relative specificity of ZIKV E, NS1, and NS5 MIA. Comparison of theresults from ZIKV E, NS1, and NS5 MIA demonstrates that antibodyresponse to NS1 and NS5 antigens is more ZIKV-specific than that to Eantigen. Specifically, ZIKV E MIA showed 100% cross-reactivity withspecimens with DENV only infection from group II, confirming thecross-reactive nature of flavivirus E antibodies. In contrast, ZIKV NS1MIA showed 14% and 22% false positive results when testing groups I andII specimens, but 100% positive accuracy when analyzing groups III andIV specimens. For ZIKV NS5 MIA, the assay exhibited 14% and 0% falsepositive results when testing groups I and II specimens, and 74% and 73%positive accuracy when analyzing groups III and IV specimens. Theresults clearly indicate that inclusion of ZIKV NS1 and NS5 in the MIAcould improve the diagnostic accuracy when compared with the MIA thatuses E protein alone.

(III) Cross reactivity between DENV/ZIKV NS1 proteins and theirantibodies. Although antibody response to ZIKV NS1 is more virus-typespecific than that to E protein (see above), we clearly observed crossreactivity between DENV and ZIKV NS1 proteins and their antibodies.Specifically, DENV NS1 MIA showed 89% and 96% positive accuracy whentesting groups II and IV specimens, respectively; and 14% and 37% falsepositive results when testing groups I and III specimens, respectively.The 37% false positive result demonstrates that DENV NS1 cross-reacts tospecimens with ZIKV-only-infection. Reciprocally, ZIKV NS1 MIA exhibited22% false positive when testing specimens with DENV-only-infection fromgroup II. Altogether, the results showed 22-37% cross reactivity betweenDENV and ZIKV NS1 proteins. The data are in agreement with the recentreport that antibodies to NS1 are largely ZIKV-specific.

Conventionally, ZIKV serologic diagnosis is mainly based on IgM-captureELISA with Emergency Use Authorization (EUA) approval from FDA. There isa need for improved methods for detection of anti-ZIKV antibodies insamples from a subject. As disclosed herein, the diagnostic power ofviral envelope protein (that elicits robust, yet cross-reactiveantibodies to other flaviviruses) is hereby combined with thedifferential power of viral nonstructural proteins NS1 and NS5 (thatinduce more virus-type specific antibodies). Disclosed herein is use ofan assay using an MIA format that can shorten assay turnaround time.Over 10,000 sera have been tested by conventional, presently FDAemergency use authorized Zika MAC-ELISA testing. Any such samplespresumptive as positive for Zika IgM according to such assay was furthertested via PRNT. Approximately 80% of the time such assays resulted inidentifying the sample as indicating “undifferentiated flavivirus”(meaning indicating that some flaviviral infection had been present, butnot definitively ruling in or out ZIKV or DENV. Further testing on over3600 of these samples permitted an identification of whether a subjecthad been infected with ZIKV, DENV, both, or neither, and whether in thepast 65 days, past 65-190 days, or more than 190 days ago. As a result,as opposed to high rates of “undifferentiated flavivirus” results, themethod disclosed herein permitted confirmatory identification of suchspecifics of infection in approximately 90% of cases where flaviviralinfection was indicated.

Furthermore, an assay disclosed herein was performed on many Zika IgMnonreactive, PCR negative specimens from pregnant women with knowndengue history. Nearly half of 199 samples from such pregnant women hadevidence of a history of Zika infection at some time according to anassay disclosed herein, combining detection of anti ZIKV E, anti-ZIKVNS1, and anti-DENV1-4 NS1 antibodies. Some pregnant women who are IgMnegative to Zika may still deliver an infant at risk of congenital Zikasyndrome, indicating the importance of accurate testing such asdisclosed herein.

Using over 3600 samples patient samples with known ZIKV and DENV PRNTresults, the method was verified and improved ability to distinguishZIKV infection from DENV infection according to methods and kits asdisclosed herein. Embodiments of a method disclosed herein are distinctfrom the single antigen-based (either E or NS1) diagnostic assays,including the E-based IgM-captured ELISA from InBios (with EUA approvalfrom FDA), NS1-based indirect ELISA from EuroImmun (approved forclinical use in Europe), and NS1-based IgM-capture ELISA from NovaTec(currently for investigational research use).

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. It will be understood that the terms “comprise”, “have”,“include”, and “contain” (and any related variants thereof) areopen-ended linking verbs. As a result, a method, step, or device that“comprises”, “has”, “includes” or “contains” one or more steps orelements possesses those one or more steps or elements, but is notlimited to possessing only those one or more steps or elements. Thecorresponding structures, materials, acts, and equivalents of all meansor step plus function elements in the claims below, if any, are intendedto include any structure, material, or act for performing the functionin combination with other claimed elements as specifically claimed. Thedisclosure herein is illustrative and not intended to be exhaustive orlimited to the invention in the form disclosed. Many modifications andvariations will be apparent to those of ordinary skill in the artwithout departing from the scope and spirit of the invention.Embodiments are described to best explain the principles of one or moreaspects of the invention and the practical application, and to enableothers of ordinary skill in the art to understand one or more aspects ofthe invention for various embodiments with various modifications as aresuited to the particular use contemplated. Further details pertaining tothe present disclosure can be found in Appendix A, attached hereto andincorporated herein in its entirety.

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1-30. (canceled)
 31. A method, comprising: (i) incubating (a) a first sample from a subject with a first plurality of populations of microspheres conjugated to viral antigens under a first condition, wherein the viral antigens conjugated to microspheres of each population of the first plurality of populations of microspheres differ from the viral antigens conjugated to microspheres of all other populations of microspheres of the first plurality of populations of microspheres, and the first condition permits high-affinity binding and low-affinity binding of antibodies in the first sample to the viral antigens, and (b) a second sample from the subject with a second plurality of populations of microspheres conjugated to viral antigens under a second condition, wherein the viral antigens conjugated to microspheres of each population of the second plurality of populations of microspheres differ from the viral antigens conjugated to microspheres of all other populations of microspheres of the second plurality of populations of microspheres, and the second condition permits high-affinity binding but not low-affinity binding of antibodies in the second sample to the viral antigens, and (ii) detecting an amount of antibodies bound to each population of the first plurality of populations of microspheres and to each population of the second plurality of populations of microspheres, wherein a first viral antigen comprises Zika virus (ZIKV) NS1, a second viral antigen comprises a flaviviral envelope protein, and a third viral antigen comprises a Dengue virus (DENV) antigen wherein the DENV antigen is selected from DENV1 NS1, DENV2 NS1, DENV3 NS1, and DENV4 NS1.
 32. The method of claim 31, wherein the third viral antigen comprises DENV1 NS1, a fourth viral antigen comprises DENV2 NS1, a fifth viral antigen comprises DENV3 NS1, and a sixth viral antigen comprises DENV4 NS1.
 33. The method of claim 31, wherein a fourth viral antigen comprises full-length ZIKV NS5.
 34. The method of claim 32, wherein a seventh viral antigen comprises full-length ZIKV NS5.
 35. The method of claim 32, wherein the flaviviral envelope protein comprises ZIKV envelope protein.
 36. The method of claim 31, wherein the second condition comprises incubating in the presence of from 7 M urea to 9 M urea.
 37. The method of claim 31, wherein the first sample, the second sample, or both samples comprise blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue, tissue homogenate, cellular extract, and spinal fluid.
 38. The method of claim 31, wherein first sample and the second sample comprise serum.
 39. The method of claim 31, wherein the subject is pregnant.
 40. The method of claim 31, wherein the subject is human.
 41. The method of claim 31, wherein the subject is a mammal.
 42. The method of claim 31, wherein the subject is a non-human primate.
 43. The method of claim 31, wherein the subject is a mouse.
 44. The method of claim 31, wherein the subject is a pig.
 45. A method, comprising: (i) incubating (a) a first sample from a subject with a first plurality of populations of microspheres conjugated to viral antigens under a first condition, wherein the viral antigens conjugated to microspheres of each population of the first plurality of populations of microspheres differ from the viral antigens conjugated to microspheres of all other populations of microspheres of the first plurality of populations of microspheres, and the first condition permits high-affinity binding and low-affinity binding of antibodies in the first sample to the viral antigens, and (b) a second sample from the subject with a second plurality of populations of microspheres conjugated to viral antigens under a second condition, wherein the viral antigens conjugated to microspheres of each population of the second plurality of populations of microspheres differ from the viral antigens conjugated to microspheres of all other populations of microspheres of the second plurality of populations of microspheres, and the second condition permits high-affinity binding but not low-affinity binding of antibodies in the second sample to the viral antigens, and (ii) detecting an amount of antibodies bound to each population of the first plurality of populations of microspheres and to each population of the second plurality of populations of microspheres, wherein a first viral antigen comprises Zika virus (ZIKV) NS1, a second viral antigen comprises a flaviviral envelope protein, a third viral antigen comprises Dengue virus (DENV) serotype 1 (DENV1) NS1, a fourth viral antigen comprises DENV2 NS1, a fifth viral antigen comprises DENV3 NS1, and a sixth viral antigen comprises DENV4 NS1.
 46. The method of claim 45, wherein a seventh viral antigen comprises full-length ZIKV NS5.
 47. The method of claim 45, wherein the flaviviral envelope protein comprises ZIKV envelope protein.
 48. The method of claim 45, wherein the subject is pregnant.
 49. The method of claim 45, wherein the subject is human.
 50. The method of claim 45, wherein the subject is a non-human primate. 